Most cellular immune responses, particularly those against intracellular parasites such as Mycobacterium tuberculosis, require the activity of CD4+ T cells. In order to stimulate antigen-specific CD4+ T cells, complexes of major histocompatibility (MHC) class II molecules bound to antigen-derived peptides must be presented on the surface of antigen presenting cells. These complexes are generated in intracellular antigen processing compartments within the late secretory and endocytic pathways. Foreign proteins access the processing compartments primarily by non-specific internalization in most antigen presenting cells, but exceptions suggest that specifically targeting antigen to these compartments via receptors greatly increases the efficiency with which antigens are processed and presented. Direct targeting and the consequent improved efficiency of antigen processing and CD4+ T cell responses is thus a consideration in development of vaccines, particularly in immunocompromised individuals. Because different compartments may contribute to antigen processing through production of distinct set of peptides, the researchers propose to target a model antigen, the 85B component of the major secreted protein product of M. tuberculosis, to distinct endosomal compartments, and monitor, by both in vitro and in vivo assays, the relative ability of differentially targeted antigen to be presented by MHC class II molecules. To do this, they will use a chimeric protein approach appending the 85B antigen to well characterized compartmental targeting signals in the context of an integral membrane protein. Chimeric proteins will be expressed in transfected cells or in mice by DNA immunization, and both biochemical and cellular aspects of antigen presentation will be assessed. The specific aims are: 1) To create chimeric cDNA expression constructs targeting the M. tuberculosis antigen 85B to distinct endosomal compartments and to verify targeting in cultured cell lines; 2) to determine biochemical and immunological consequences of 85B targeting to distinct endosomal compartments for antigen processing and presentation in cultured human B lymphoblastoid cell lines; and 3) to determine the consequences of differential targeting for 85B-specific immune responses in mice immunized with DNA.